Certain products and by-products of naturally occurring metabolic processes in microbial cells or in the cells of animals and, advantageously plants, have utility for a wide range of industries, including the feed, food, cosmetics and pharmaceutical industries. These molecules, which are collectively termed “fine chemicals”, also include, for example, lipids and fatty acids, one representative class of which are the polyunsaturated fatty acids. Polyunsaturated fatty acids (PUFAs) are added for example to infant formula for increasing the nutritional value of these foods. PUFAs have, for example, a positive effect on the cholesterol level in the blood of humans and are therefore useful for protection against heart disease. Fine chemicals such as polyunsaturated fatty acids (PUFAs) can be isolated from animal sources such as, for example, fish, or produced by microorganisms by culturing microorganisms which have been developed such that they produce and accumulate or secrete large amounts of one or more desired molecules.
Fatty acids and triglycerides have a multiplicity of uses in the food industry, in animal nutrition, in cosmetics and in the pharmacological sector. Depending on whether they take the form of free saturated or unsaturated fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a variety of uses. Polyunsaturated Ω3-fatty acids and Ω6-fatty acids constitute an important part of animal and human nutrition. Owing to the present-day composition of human nutrition, an addition of polyunsaturated Ω3-fatty acids, which are predominantly found in fish oils, to the food is of particular importance. Thus, for example, polyunsaturated fatty acids such as docosahexaenoic acid (=DHA, C22:6Δ4,7,10,13,16,19) or eisosapentaenoic acid (=EPA, C20:5Δ5,8,11,14,17) is added to baby formula for increasing the nutritional value. DHA is said to have a positive effect on brain development.
The various acids and triglycerides are obtained mainly from microorganisms such as Mortierella or from oil-producing plants such as soybeans, oilseed rape, sunflower, algae such as Cryptocodinium or Phaeodactylum and others, the products being obtained, as a rule, in the form of their triacylglycerides (=triglycerides=triglycerols). However, they can also be obtained from animals such as, for example, fish. The free fatty acids are advantageously prepared by hydrolysis. Higher polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (=ARA, C20:4Δ5,8,11,14), dihomo-γ-linolenic acid (C20:3Δ8,11,14) or docosapentaenoic acid (DPA, C22:5Δ7,10,13,16,19) cannot be isolated from oil crops such as oilseed rape, soybeans, sunflower, safflower or others. Conventional natural sources of these fatty acids are fish such as herring, salmon, sardine, redfish, eel, carp, trout, halibut, mackerel, zander or tuna, or algae.
Depending on the intended purpose, oils with saturated or with unsaturated fatty acids are preferred; thus, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition. The polyunsaturated Ω3-fatty acids are said to have a positive effect on the cholesterol level in the blood and thus on the possibility of preventing heart disease. The risk of heart disease, stroke or hypertension can be reduced markedly by adding these ω3-fatty acids to the food. Also, Ω3-fatty acids can have a positive effect on inflammatory processes, specifically chronically inflammatory processes in connection with immunological diseases such as rheumatoid arthritis. These fatty acids are therefore added to foodstuffs, specifically dietetic foodstuffs, or are used in medicaments.
In connection with these rheumatic diseases due to the usual composition of our foods, Ω6-fatty acids such as arachidonic acid tend to have a negative effect on these diseases.
Ω3- and Ω6-fatty acids are precursors of tissue hormones, what are known as eicosanoids such as the postaglandins, which are derived from dihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, the thromoxanes and the leukotrienes, which are derived from arachidonic acid and eicosapentaenoic acid. Eicosanoids (known as the PG2 series), which are formed from Ω6-fatty acids, promote, as a rule, inflammatory reactions, while eicosanoids (known as the PG3 series) from Ω3-fatty acids have a minor, or no, proinflammatory action.
Owing to the positive properties, there has been no lack of attempts in the past to make available genes which are involved in the synthesis of fatty acids or triglycerides, for the production, in various organisms, of oils with a modified content of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. A Δ15-desaturase is claimed in WO 93/11245 and a Δ12-desaturase is claimed in WO 94/11516. Further desaturases are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. However, the biochemical characterization of the various desaturases is incomplete as yet since the enzymes, being membrane-bound proteins, can only be isolated and characterized with great difficulty (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et-al., Plant Physiol. Biochem., 26, 1988: 777-792). As a rule, membrane-bound desaturases are characterized by introduction into a suitable organism which is subsequently analyzed for enzyme activity by means of analyses of the starting material and the product. Δ6-Desaturases are described in WO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No. 5,614,393, WO 96/21022, WO00/21557 and WO 99/27111, and their application for the production in transgenic organisms has also been described, such as in WO98/46763 WO98/46764, WO9846765. In this context, the expression of various desaturases is also described and claimed, as is the case in WO99/64616 or WO98/46776, as is the formation of polyunsaturated fatty acids. As regards the efficacy of the expression of desaturases and their effect on the formation of polyunsaturated fatty acids, it must be noted that only minor contents of Δ6-unsaturated fatty acids/lipids, such as, for example, gamma-linolenic acid and stearidonic acid, have been obtained by expression of a single desaturase, as described to date. Moreover, a mixture of ω3- and ω6-fatty acids has been obtained as a rule, since all of the Δ6-desaturases described to date converted for example not only linoleic acid (ω6-fatty acid), but also α-linolenic acid (ω3-fatty acid).
Particularly suitable microorganisms for the production of PUFAs are microorganisms such as Thraustochytrium species or Schizochytrium species, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor. Strain selection has made possible the development of mutant strains of the microorganisms in question which produce a series of desirable compounds, including PUFAs. The mutation and selection of strains with an improved production of a particular molecule, such as the polyunsaturated fatty acids, is, however, a time-consuming and difficult procedure. This is why recombinant methods are preferred whenever possible, as described above. However, only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or APA can be produced with the aid of the abovementioned microorganisms, these unsaturated fatty acids being obtained, as a rule, as fatty acid mixtures of, for example, EPA, DPA and DHA, depending on the microorganism used.
As an alternative, the production of fine chemicals can suitably be carried out on a large scale via the production in plants which have been developed such that they produce the abovementioned PUFAs. Plants which are particularly suited to this purpose are oil crops, which comprise large amounts of lipid compounds, such as oilseed rape, canola, linseed, soyabeans, sunflowers, borage and evening primrose. However, other crop plants which comprise oils or lipids and fatty acids are also well suited, as mentioned in the extensive description of the present invention. Conventional breeding has given rise to a series of mutant plants which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of new plant varieties with improved production of a particular molecule is a time-consuming and difficult procedure or is indeed impossible if the compound does not occur naturally in the plant in question, as in the case of polyunsaturated C18-, C20-fatty acids and C22-fatty acids and those with longer carbon chains.
Owing to the positive properties of unsaturated fatty acids, there has been no lack of attempts in the past to make available these genes which are involved in the synthesis of fatty acids or triglycerides for the production, in various plants, of oils with a modified content of polyunsaturated fatty acids. However, it has been impossible as yet to produce longer-chain polyunsaturated C20- and/or C22-fatty acids such as EPA or ARA in plants.